The CFTR protein exposes only ˜3% of its mass on the exterior surface of the plasma membrane making its detection difficult without permeabilization of the membrane. After the CFTR amino acid sequence and predicted 2D membrane topology became available (Riordan et al., 1989) extensive efforts were made to raise antibodies that would recognize epitopes within the extracytoplasmic loops (ELs) and some success was reported (Denning et al., 1992). However, both this study, where polyclonal antisera were generated against a synthetic peptide corresponding to the sequence of the first EL, and other work at Transgene S.A. where monoclonal antibodies were generated against a similar peptide, were never confirmed or shown to be of practical use in the ensuing decade. Efforts in several other laboratories to generate EL1 antibodies capable of detecting CFTR from the cell exterior were unsuccessful.
A somewhat more successful approach was taken by Howard and coworkers (Howard et al., 1995; Howard et al., 1996; Schultz et al., 1997) who inserted exogenous epitopes in the fourth extracytoplasmic loop (EL4). However in so doing the native N-glycosylation sites at asparagine residues 894 and 900 were removed so that only unglycosylated CFTR could be detected at the cell surface. Although it is known that unglycosylated CFTR is transported to the cell surface where it has some chloride channel activity, it is also known that it is expressed poorly compared to the native glycosylated molecule and has a much shorter lifetime. Therefore it is not useful to monitor conditions that would favor transport of the ΔF508 CFTR polypeptide to the cell surface. Introduction of an epitope into EL4 has also been referred to in some more recent publications (Konstas et al., 2002; Benharouga et al., 2003). However, no details of how this was done were provided and results were shown only for Xenopus oocytes and not in a cell system amenable to high throughput screening (Konstas et al., 2002). It is not known whether or not this EL4 epitope insertion enables only detection of unglycosylated CFTR, as was the case with the earlier EL4 insertion (Howard et al., 1995).
Approximately 90% of CF patients have the ΔF508 mutation at at least one CFTR allele (Drumm and Collins, 1993). The deletion of this single amino acid prevents the nascent protein from maturing conformationally and being transported to its site of action at the cell surface plasma membrane (reviewed in Riordan, 1999; Kopito, 1999; Gelman and Kopito, 2002). The latter article emphasizes that the search for small molecules that either circumvent or overcome this defect is a vitally important approach to the discovery of new therapeutics for the disease.